Chemical modifications of protein molecules usually involve reactions with specific amino acids such as lysine or tyrosine. However, such modifications are likely to damage the biological function, activity or specificity of the protein if the modifications occur at biologically active sites. Alteration of a protein's biological function(s) which results from chemical modification of the protein depends on the number and location of modified amino acids within its allosteric and/or active sites.
The development of monoclonal antibodies through hybridoma technology has made possible the production and isolation of large quantities of homogeneous antibody to defined antigenic determinants such as, for example, membrane surface features unique to tumor tissue. Thus, linking of radionuclides, toxins, chemotherapeutic agents, or fluorescent agents or compounds to monoclonal antibodies against antigenic determinants associated with a particular tumor is desirable for tumor imaging or therapy.
Antibodies in general, and monoclonal antibodies (MAb) in particular, are members of the class of complex proteins known as immunoglobulins and have a basic structure of two identical light and two identical heavy polypeptide chains joined together by interchain disulphide bonds in the form of a Y-shape as seen in FIG. 1. Treatment of an antibody with papain cleaves the molecule into three fragments or regions, two of which fragments bind to antigens (Fab) and one fragment which is readily crystallizable (Fc). The amino acid sequence of the N-terminal end of each of the chains varies greatly between molecules and is thus known as the variable region. The Variable regions of the two heavy and light chains are denoted V.sub.H and V.sub.L respectively. Each variable region of each chain also contains three hypervariable regions which are more variable than other regions in terms of amino acid substitutions, deletions, and insertions. The V.sub.H and V.sub.L domains are folded in such a way that the hypervariable regions are brought together to form an antigen binding site. The specificity of the antigen binding site is determined by the amino acid sequence of both the heavy and light chains. All antibody molecules, except for those of the IgM class, have two identical antigen binding sites.
As is well known in the art, one of the more preferred ways to attach a drug, toxin or radiolabeled compound to an immunoglobulin, such as a monoclonal antibody, is via a covalent linkage to the lysine residues of the immunoglobulin. See. Koppel, G. A., Bioconjugate Chem. 1:13-23 (1990), the disclosure of which is incorporated herein by reference. A major drawback to this method of protein modification is that the epsilon-amino group of lysines in the Fab portion will be altered, and if one of such group is essential for the binding to a particular antigen, the immunoreactivity of the monoclonal antibody will be degraded, if not completely lost. In such an instance the resulting drug, toxin, or radiolabeled monoclonal antibody conjugate is rendered highly ineffective, if not useless.
Previous methods of selective modification of immunoglobulins have been limited because they have been directed to modification of those immunoglobulins with sugar residues attached to the Fc region.
It is therefore desirable to modify intact monoclonal antibodies at specific sites to allow for the coupling of diagnostic or therapeutic agents without significantly diminishing immunoreactivity.
It is also desirable to modify monoclonal antibodies at specific sites regardless of whether the antibody has sugar residues.
In the following description of the present invention, the term "biodistribution" refers to the distribution of antibody in a subject to which antibody has been administered. By contrast, "uptake" refers to the quantity of antibody in a given tissue.